The present invention relates to methods for detecting the presence of ribonuclease enzymes, more specifically to methods that provide for a visual detection assay. The present invention further provides novel nucleic acid compositions used as substrates for such assays and encompasses kits for performing the methods of the invention.
Ribonuclease (RNase) enzymes degrade polymeric ribonucleic acids (RNA) into shorter fragments or component nucleotides. All organisms produce ribonucleases and these enzymes are found in most environments. The properties of a number of ribonucleases are described by D""Alessio and Riordan (1997. As a group, most ribonucleases are specific for single-stranded RNA and will not cleave RNA in duplex form. Further, ribonucleases generally cleave at the 3xe2x80x2-end of a ribonucleic acid phosphodiester linkage. Many different RNase enzymes exist, some of which have little or no substrate preference while others are sequence specific. For example, ribonuclease I, from E. coli, is a non-specific endoribonuclease that degrades RNA by cleavage at any base. Ribonuclease A, from mammalian pancreas, is a base-specific endoribonuclease that degrades RNA by cleavage following a pyrimidine (uridine or cytosine) base. Ribonuclease T1, from Aspergillus oryzae, is a base-specific endoribonuclease that degrades RNA by cleavage following a guanosine residue. These three RNase enzymes are noteworthy in that they are routinely employed in standard molecular biology protocols to remove unwanted RNA from samples or as a component in certain assay procedures.
Single-strand specific RNases are the primary nuclease activity encountered in research laboratories as an unwanted contaminant. Double-strand specific RNases have been described, however these are rare and not routinely found in most laboratory settings. RNase H cleaves RNA only when complexed as a heteroduplex with DNA and is not of concern as a laboratory contaminant.
Ribonucleases are present in all laboratories as ubiquitous environmental contaminants. RNases are also found in most molecular biology laboratories as purified enzyme stocks. In laboratories that study RNA, careful attention to experimental protocol is needed to avoid contamination of reagents with RNases; for example, gloves must be worn at all times to prevent contact with the RNases that are universally present on human skin. Regardless of source, the presence of a contaminant RNase will degrade any RNA that comes in contact with that reagent, resulting in the loss of valuable samples or interfering with time-consuming experiments. Once present, removing RNase activity from a laboratory reagent is difficult. Most RNase enzymes are remarkably stable and survive harsh treatments that are routinely used to eliminate other unwanted biologic activities, such as autoclaving. Methods that remove RNase activity range from baking glassware at very high temperature to treating reagent stocks with the highly toxic chemical diethylpyrocarbonate (Sambrook et al., 1989). In spite of such attention, RNase contamination remains a chronic problem and monitoring for the presence of RNase activity is a routine quality control (QC) step in most research and industrial laboratories. As such, methods are needed that would detect the presence of RNase activities commonly encountered in the laboratory setting and that are suitable for routine, frequent use.
Many methods have been devised attempting to measure RNase activity. RNase assays can be grossly divided into methods that detect degradation of heterogeneous RNA obtained from biological sources and methods that detect specific cleavage of a well-defined synthetic substrate, such as an oligonucleotide. In general, use of a synthetic substrate affords both increased sensitivity and improved specificity. Many different detection modalities have been incorporated into these assays, including direct staining, spectrophotometric and colorimetric readouts, chromogenic cascade, radioactive tracer, fluorescence polarization, and fluorescence quenching methods.
Choice of detection method will affect assay sensitivity and ease of use. For use in determining the presence or absence of RNase contamination in laboratory reagents, the method should be sufficiently sensitive to detect the presence of RNase enzymes at the lowest level that will degrade experimental samples in actual use. An insensitive assay would xe2x80x9cpassxe2x80x9d reagents that are contaminated, which is undesirable. Conversely, an assay could be too sensitive and might xe2x80x9cfailxe2x80x9d reagents that, from a practical standpoint, are not contaminated and would therefore also be undesirable.
A detection limit within the range of 1-100 picogram/ml (pg/ml) of RNase A is ideal for a reagent QC assay. Commercial assays currently available are sensitive in the 10-100 pg/ml range (Ambion Catalog, 1999). Since such an assay would be used repeatedly, it is also desirable that the method be rapid and easy to perform. Preferably, such an assay could be done at the site of suspected contamination and offer a rapid visual readout.
The original unit definition of ribonuclease activity is based upon the method of Kunitz (1946) which employs a spectrophotometric assay to measure the decrease in absorbance at 300 nm that occurs with degradation of heterogeneous RNA. While the method has been improved (Oshima, 1976), it is insensitive and therefore of little use as a quality control (QC) assay.
Another method to detect RNase activity involves separation and assay of component enzyme activities within a sample using polyacrylamide gel electrophoresis (Wilson, 1969). RNase enzymes can be detected in the acrylamide matrix by direct staining or by incubation with a heterogeneous substrate RNA and an RNA staining dye, such as toluidine blue. While conceptually simple, this approach is time-consuming and relatively insensitive, having a lower limit of detection of about 1 unit of RNase I. In an improvement of this technique, Karpetsky (1980) describes a polynucleotide/polyacrylamide-gel electrophoresis method that improves sensitivity to below 100 pg of RNase A. However, even the improved method remains slow and cumbersome and is better suited to the analysis of ribonuclease activities in biologic specimens than to the QC of laboratory reagents.
Another approach to detect RNase activity is described by Egly and Kempf (1976). This procedure detects release of soluble 125Iodine-labeled RNA from an insoluble RNA-agarose matrix in the presence of ribonuclease. The method is capable of detecting the presence of RNase A at levels as low as 0.01 pg/ml and is actually too sensitive for use as a routine QC assay. Furthermore, this method employs a hazardous radioactive isotope as reporter that is not desirable for use in most laboratory or industrial settings.
Another approach to detect RNase activity is described by Wagner (1983). RNA forms a complex with Pyronine-Y that has an optical absorbance maximum at 572 nm. Degradation of high molecular weight RNA by ribonuclease activity results in loss of absorbance at 572 nm in a linear and quantitative fashion. The method, however, is only capable of detecting about 2 ng/ml RNase A in a test sample and has insufficient sensitivity for use as a QC assay.
Another approach to detect RNase activity was described by Greiner-Stoeffele (1996). The dye methylene blue intercalates into high molecular weight ribonucleic acid forming a dye-RNA complex. Upon degradation by ribonuclease action, methylene blue is released and absorbance at 688 nm decreases. This method, however, is also relatively insensitive and can detect ribonuclease activity only down to about 25 ng/ml, which is inadequate for use as a QC assay.
Another approach to detect RNase activity is described by Karn (1979). Ribonuclease A-mediated cleavage of a synthetic ribonucleotide dimer substrate was detected by a cascade of enzymatic reactions involving adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase that ultimately forms a detectable blue tetrazolium salt. The method can detect the presence of 0.066 units of RNase A (about 100 ng), insufficient for use as a QC assay. Furthermore, the procedure is lengthy, complex and requires modification to detect the presence of ribonucleases other than RNase A.
Another approach to detect RNase activity is described by Witmer (1991). A synthetic ribonucleotide substrate, U-3xe2x80x2-BCIP, was synthesized that releases a reporter group in the presence of RNase A that could be detected spectrophotometrically by absorbance at 650 nm. While this chromogenic method is simple to use, it is insensitive and is better suited for applications such as the in vivo bacterial colony assays taught by Witmer than for use as a reagent QC assay.
Fluorescence-quenching detection is used in many applications in the biological sciences; representative examples include methods to detect proteolytic enzyme activity (Yaron, 1979), methods to detect DNA restriction endonuclease activity (Ghosh, 1994), methods to detect the 5xe2x80x2-nuclease activity of DNA polymerase (Gelfand, 1993), methods to detect nucleic acid sequence identity (Gelfand, 1993; Tyagi, 1999; Livak, 1999; Nazarenko, 1999; Nadeau, 1999), and methods to detect bimolecular protein interactions in an immunoassay (Maggio, 1980). A synthetic oligoribonucleotide having a Fam reporter group and a Tamra quencher group has been used as a FRET probe to detect hammerhead ribozyme activity (Hanne, 1998). Fluorescence Resonance Energy Transfer (FRET) and fluorescence quenching methods are reviewed by Morrison (1992).
Zelenko (1994) describes synthesis of a dinucleotide substrate uridylyl-3xe2x80x25xe2x80x2-deoxyadenosine that is conjugated to a fluorophore (O-aminobenzoic acid) on one end and a fluorescence quencher (2,4-nitroaniline) on the opposite end of the molecule. Cleavage by RNase A separates the fluorophore and quencher, leading to a detectable increase in fluorescence. The substrate was designed specifically for use in kinetic studies of RNase A activity and will react only with the subset of ribonuclease enzymes that cleave at a uracil residue. Having a limited spectrum of sensitivity, this reagent is not suitable for use as a single substrate in an RNase QC assay.
James (1998) describe an alternative substrate for kinetic studies of RNase A in which a 9-mer chimeric oligonucleotide that contains a single ribonucleotide uracil base flanked by deoxyadenosine residues was modified with a 5xe2x80x2 fluorescein reporter group and a 3xe2x80x2 rhodamine quencher group. The utility of the substrate is limited in that it can detect only those ribonucleases that cleave at a uracil residue. Further, assay results must be detected using a fluorometer due to background fluorescence of the rhodamine quencher group.
Kelemen (1999) describes a similar substrate having somewhat greater sensitivity measuring RNase A kinetics with the following composition: SEQ ID. NO:2: 5xe2x80x2 Fluorescein-AuAA-Tamra 3xe2x80x2. Like the James reagent, the Kelemen substrate is limited to detecting ribonucleases that cleave at a uracil residue and requires the use of a fluorometer.
James (1998) and Kelemen (1999), therefore, have described use of fluorescent-labeled oligonucleotide probes with FRET/quenching to study the catalytic properties of RNase A. Both compositions are chimeric DNA-RNA oligonucleotides that contain a single internal uridine base, use a fluorescein dye as reporter group, and use a quencher group that is a fluorophore that itself emits light in the visible spectrum, so methods that use these substrates require availability of a fluorometer for detection. These probes were optimized for kinetic studies of RNase A and cannot be used to detect the presence of RNase enzymes that do not cleave at a uridine residue. In addition, both compositions include DNA residues, which are subject to cleavage by DNase enzymes, so cleavage is not RNase specific. They are, therefore, not useful as a tool to assay for the presence of contaminating RNase activity.
Burke (1998) describes a method that utilizes fluorescence polarization detection techniques to measure cleavage of short, synthetic nucleic acid probes. A commercial kit for performing RNase detection of Burke is available (Pan Vera Catalog, 2000). Wilson (2000) describes a variant of this technique that examines real-time degradation of a long, synthetic RNA species (made by in vitro transcription) using fluorescence anisotropy. The fluorescence polarization-based techniques that must be employed, however, cannot be performed without a specialized fluorescence polarization fluorometer, which is not available in most laboratories.
Another commercial kit for the detection of RNase activity measures the release of soluble fluorescent dye from a precipitated (i.e., insoluble) fluorescent RNA substrate (PanVera Catalog, 2000). This method is less sensitive than the fluorescence polarization method and also requires availability of a fluorometer, thereby limiting the utility of the assay.
A commercial kit is available that uses a biotin-labeled RNA substrate immobilized on dipsticks to test for the presence of RNase activity (Ambion Catalog, 1999). Detection is achieved using a visual calorimetric method. In the absence of RNase, the substrate remains intact and the colorimetric assay develops a blue spot on the dipstick while in the presence of RNase the label is cleaved and no color develops. This assay is labor intensive, takes over 3 hours to perform, and is not well suited for high-throughput QC use.
Another commercial RNase detection kit employs gel electrophoresis to visualize degradation of a high molecular weight RNA in the presence of ribonuclease activity (Mo Bio, Web Catalog, 2000). The method is a multi-step, labor intensive protocol that is very expensive, making it unsuitable for routine QC use.
It is apparent from the above discussion that, while progress has been made in methodology to detect ribonuclease activity, existing assays have significant limitations. None are suitable for use as a universal ribonuclease detection system (i.e., a QC assay). A ribonuclease detection method suitable for use in a QC assay should meet the following 7 criteria:
1) The assay will be highly sensitive.
2) The assay will be highly specific.
3) The assay will detect a broad spectrum of ribonuclease activities.
4) The assay reagent(s) will be inexpensive and suitable for commercial manufacture.
5) The assay method will be both simple and rapid.
6) The assay method will allow for visual detection and will not require the use of highly specialized equipment.
7) The assay will not employ any hazardous compounds.
Clearly new methods, or improvements in earlier methods, are needed. In particular, a need exists for an RNase assay that is rapid, sensitive, within the desired range and allows for visual detection.
The present invention describes novel nucleic acid compositions and methods for a fluorescence-quenching based assay of ribonuclease activity that overcomes the deficiencies of earlier teachings and is suitable for use as a research or industrial quality control assay. The method is highly sensitive, highly specific, capable of detecting a broad spectrum of ribonuclease enzymes, employs reagents that can be manufactured using commercial reagents, is rapid and easy to perform, does not use any hazardous reagents, and can be performed without any specialized equipment Further, the method provides for a visual assay format. The visual assay is sensitive to 10 pg/ml RNase A, a level that is suitable for use as a QC assay. Surprisingly, sensitivity of the visual assay is comparable to that of existing commercial assays which require use of a fluorometer for detection. Compositions of the invention can also be used with fluorometric detection and are compatible with automated high-throughput robotic systems as are commonly employed in industrial settings.
The present invention further relates to novel nucleic acid compositions useful in the practice of such techniques and, still further, to kits for performing the method of the invention.
The present invention relates to methods for detecting ribonuclease activity in a sample, comprising: 1) incubating a of a synthetic Substrate or mixture of Substrates in the sample, for a time sufficient for cleavage of the Substrates(s) by a ribonuclease enzyme, wherein said Substrate(s) comprises a single-stranded nucleic acid molecule containing at least one ribonucleotide residue at an internal position that functions as a cleavage site, a fluorescence reporter group on one side of the cleavage sites, and a fluorescence-quenching group on the other side of the cleavage site, and 2) visual detection of a fluorescence signal, wherein detection of a fluorescence signal indicates that a ribonuclease cleavage event has occurred, and, therefore, the sample contains ribonuclease activity. The compositions of the invention are also compatible with other detection modalities (e.g., fluorometry).
The Substrate oligonucleotide of the invention comprises a fluorescent reporter group and a quencher group in such physical proximity that the fluorescence signal from the reporter group is suppressed by the quencher group. Cleavage of the Substrate with a ribonuclease enzyme leads to strand cleavage and physical separation of the reporter group from the quencher group. Separation of reporter and quencher eliminates quenching, resulting in an increase in fluorescence emission from the reporter group. When the quencher is a so-called xe2x80x9cdark quencherxe2x80x9d, the resulting fluorescence signal can be detected by direct visual inspection. Cleavage of the Substrate compositions described in the present invention can also be detected by fluorometry.
In one embodiment, the synthetic Substrate is an oligonucleotide comprising ribonucleotide residues. The synthetic Substrate can also be a chimeric oligonucleotide comprising RNase-cleavable, e.g., RNA, residues, modified RNase-resistant RNA residues, or modified DNA residues that are resistant to cleavage by deoxyribonucleases. Substrate composition is such that cleavage is a ribonuclease-specific event and that cleavage by deoxyribonucleases does not occur.
In a preferred embodiment, the synthetic Substrate is a chimeric oligonucleotide comprising ribonucleotide residue(s) and modified ribonucleotide residue(s). In a more preferred embodiment, the synthetic Substrate is a chimeric oligonucleotide comprising ribonucleotide residues and 2xe2x80x2-O-methyl ribonucleotide residues. In a most preferred embodiment, the synthetic Substrate is a chimeric oligonucleotide comprising 2xe2x80x2-O-methyl ribonucleotide residues and one or more of each of the four ribonucleotide residues, adenosine, cytosine, guanosine, and uridine. Inclusion of the four distinct ribonucleotide bases in a single Substrate allows for detection of an increased spectrum of ribonuclease enzyme activities by a single Substrate oligonucleotide.
To enable visual detection methods, the quenching group is itself not capable of fluorescence emission, being a xe2x80x9cdark quencherxe2x80x9d. Use of a xe2x80x9cdark quencherxe2x80x9d eliminates the background fluorescence of the intact Substrate that would otherwise occur as a result of energy transfer from the reporter fluorophore. In one preferred embodiment, the fluorescence quencher comprises dabcyl (4-(4xe2x80x2-dimethylaminophenylazo)benzoic acid). In a most preferred embodiment, the fluorescence quencher is comprised of QSY(trademark)-7 carboxylic acid, succinimidyl ester (N,Nxe2x80x2-dimethyl-N,Nxe2x80x2-diphenyl-4-((5-t-butoxycarbonylaminopentyl)aminocarbonyl) piperidinylsulfonerhodamine; a diarylrhodamine derivative from Molecular Probes, Eugene, Oreg.). Any suitable fluorophore may be used as reporter provided its spectral properties are favorable for use with the chosen quencher. A variety of fluorophores can be used as reporters, including but not limited to, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, rhodamine, tetramethylrhodamine, Cy-dyes, Texas Red, Bodipy dyes, and Alexa dyes.
The method of the invention proceeds in two steps. First, the test sample is mixed with the Substrate reagent and incubated. Substrate can be mixed alone with the test sample or, more preferably, will be mixed with an appropriate buffer, e.g., one of a composition as described herein. Second, visual detection of fluorescence is performed. As fluorescence above background indicates fluorescence emission of the reaction product, i.e. the cleaved Substrate, detection of such fluorescence indicates that RNase activity is present in the test sample. The method provides that this step can be done with unassisted visual inspection. In particular, visual detection can be performed using a standard ultraviolet (UV) light source of the kind found in most molecular biology laboratories to provide fluorescence excitation. Substrates of the invention can also be utilized in assay formats in which detection of Substrate cleavage is done using a multi-well fluorescence plate reader or a tube fluorometer.
The present invention further features kits for detecting ribonuclease activity comprising a Substrate nucleic acid(s) and instructions for use. Such kits may optionally contain one or more of: a positive control ribonuclease, RNase-free water, and a buffer. It is also provided that said kits may include RNase-free laboratory plasticware, for example, thin-walled, UV transparent microtubes for use with the visual detection method and/or multiwell plates for use with plate-fluorometer detection methods in a high-throughput format.
Accordingly, the present invention provides a method for detecting ribonuclease activity in a test sample, comprising: (a) contacting the test sample with a substrate, thereby creating a test reaction mixture, wherein said substrate comprises a nucleic acid molecule comprising (i) a cleavage domain comprising a single-stranded region, said single-stranded region comprising at least one internucleotide linkage; (ii) a fluorescence reporter group on one side of the internucleotide linkage; and (iii) a non-fluorescent fluorescence-quenching group on the other side of the internucleotide linkage; (b) incubating said test reaction mixture for a time sufficient for cleavage of the substrate by a ribonuclease in the sample; and (c) determining whether a visually detectable fluorescence signal is emitted from the test reaction mixture, wherein emission of a fluorescence signal from the reaction mixture indicates that the sample contains ribonuclease activity.
While the methods of the invention can be practiced without the use of a control sample, in certain embodiments of the invention it is desirable to assay in parallel with the test sample a control sample comprising a known amount of RNase activity. Where the control sample is used as a negative control, the control sample preferably contains no detectable RNase activity. Thus, the present invention further provides a method for detecting ribonuclease activity in a test sample, comprising: (a) contacting the test sample with a substrate, thereby creating a test reaction mixture, wherein said substrate comprises a nucleic acid molecule comprising: (i) a cleavage domain comprising a single-stranded region, said single-stranded region comprising at least one internucleotide linkage; (ii) a fluorescence reporter group on one side of the internucleotide linkage; and (iii) a non-fluorescent fluorescence-quenching group on the other side of the internucleotide linkage; (b) incubating said test reaction mixture for a time sufficient for cleavage of the substrate by a ribonuclease activity in the test sample; (c) determining whether a visually detectable fluorescence signal is emitted from the test reaction mixture; (d) contacting a control sample with the substrate, said control sample comprising a predetermined amount of ribonuclease, thereby creating a control reaction mixture; (e) incubating said control reaction mixture for a time sufficient for cleavage of the substrate by a ribonuclease in the control sample; (f) determining whether a visually detectable fluorescence signal is emitted from the control reaction mixture; wherein detection of a greater fluorescence signal in the test reaction mixture than in the control reaction mixture indicates that the test sample contains greater ribonuclease activity than in the control sample, and wherein detection of a lesser fluorescence signal in the test reaction mixture than in the control reaction mixture indicates that the test sample contains less ribonuclease activity than in the control sample. In one embodiment, the predetermined amount of ribonuclease is no ribonuclease, such that detection of a greater fluorescence signal in the test reaction mixture than in the control reaction mixture indicates that the test sample contains ribonuclease activity.
The methods of the invention can further entail contacting the test sample with a buffer before or during step (a).
As stated above, the present invention further provides compositions and kits for practicing the present methods. Thus, in certain embodiments, the present invention provides a nucleic acid comprising: (a) a cleavage domain comprising a single-stranded region, said single-stranded region comprising at least one internucleotide linkage; (b) a fluorescence reporter group on one side of the internucleotide linkage; and (c) a non-fluorescent fluorescence-quenching group on the other side of the internucleotide linkage. In other embodiments, the present invention provides a kit comprising: (a) in one container, a substrate, said substrate comprising a nucleic acid molecule comprising a single stranded region, said single-stranded region comprising: (i) a cleavage domain comprising a single-stranded region, said single-stranded region comprising at least one internucleotide linkage 3xe2x80x2 to an adenosine residue, at least one internucleotide linkage 3xe2x80x2 to a cytosine residue, at least one internucleotide linkage 3xe2x80x2 to a guanosine residue, and at least one internucleotide linkage 3xe2x80x2 to a uridine residue, and wherein said cleavage domain does not comprise a deoxyribonuclease-cleavable internucleotide linkage; (ii) a fluorescence reporter group on one side of the internucleotide linkages; and (iii) a non-fluorescent fluorescence-quenching group on the other side of the internucleotide linkages.
In a preferred embodiment of the foregoing methods and compositions, the single stranded region of the cleavage domain comprises at least on internucleotide linkage 3xe2x80x2 to an adenosine residue, at least one internucleotide linkage 3xe2x80x2 to a cytosine residue, at least one internucleotide linkage 3xe2x80x2 to a guanosine residue, and at least one internucleotide linkage 3xe2x80x2 to a uridine residue. In another preferred embodiment, the cleavage domain does not comprise a deoxyribonuclease-cleavable internucleotide linkage. In yet another referred embodiment, the single stranded region of the cleavage domain comprises at least on internucleotide linkage 3xe2x80x2 to an adenosine residue, at least one internucleotide linkage 3xe2x80x2 to a cytosine residue, at least one internucleotide linkage 3xe2x80x2 to a guanosine residue, and at least one internucleotide linkage 3xe2x80x2 to a uridine residue and the cleavage domain does not comprise a deoxyribonuclease-cleavable internucleotide linkage.
With respect to the fluorescence quenching group, any compound that is a dark quencher can be used in the methods and compositions of the invention. Numerous compounds are capable of fluorescence quenching, many of which are not themselves fluorescent, i.e., are dark quenchers.) In one embodiment, the fluorescence-quenching group is a nitrogen-substituted xanthene compound, a substituted 4-(phenyldiazenyl)phenylamine compound, or a substituted 4-(phenyldiazenyl)naphthylamine compound. In certain specific modes of the embodiment, the fluorescence-quenching group is 4-(4xe2x80x2-dimethylaminophenylazo)benzoic acid), N,Nxe2x80x2-dimethyl-N,Nxe2x80x2-diphenyl-4-((5-t-butoxycarbonylaminopentyl) aminocarbonyl) piperidinylsulfonerhodamine (sold as QSY-7(trademark) by Molecular Probes, Eugene, Oreg.), 4xe2x80x2,5xe2x80x2-dinitrofluorescein, pipecolic acid amide (sold as QSY-33(trademark) by Molecular Probes, Eugene, Oreg.) 4-[4-nitrophenyldiazinyl]phenylamine, or 4-[4-nitrophenyldiazinyl]naphthylamine (sold by Epoch Biosciences, Bothell, Wash.). In other specific modes of the embodiment, the fluorescence-quenching group is Black-Hole Quenchers(trademark) 1, 2, or 3 (Biosearch Technologies, Inc.).
In certain embodiments, the fluorescence reporter group is fluorescein, tetrachlorofluorescein, hexachlorofluorescein, rhodamine, tetramethylrhodamine, a Cy dye, Texas Red, a Bodipy dye, or an Alexa dye.
With respect to the foregoing methods and compositions, the fluorescence reporter group or the fluorescence quenching group can be, but is not necessarily, attached to the 5xe2x80x2-terminal nucleotide of the substrate.
The nucleic acids of the invention, including those for use as substrates in the methods of the invention, are preferably single-stranded RNA molecule. In other embodiments, the nucleic acids of the invention are chimeric oligonucleotides comprising a nuclease resistant modified ribonucleotide residue. Exemplary RNase resistant modified ribonucleotide residues include 2xe2x80x2-O-methyl ribonucleotides, 2xe2x80x2-methoxyethoxy ribonucleotides, 2xe2x80x2-O-allyl ribonucleotides, 2xe2x80x2-O-pentyl ribonucleotides, and 2xe2x80x2-O-butyl ribonucleotides. In a preferred mode of the embodiment, the modified ribonucleotide residue is at the 5xe2x80x2-terminus or the 3xe2x80x2-terminus of the cleavage domain. In yet other embodiments, the nucleic acids of the invention are chimeric oligonucleotides comprising a deoxyribonuclease resistant modified deoxyribonucleotide residue. In specific modes of the embodiments, the deoxyribonuclease resistant modified deoxyribonucleotide residue is a phosphotriester deoxyribonucleotide, a methylphosphonate deoxyribonucleotide, a phosphoramidate deoxyribonucleotide, a phosphorothioate deoxyribonucleotide, a phosphorodithioate deoxyribonucleotide, or a boranophosphate deoxyribonucleotide. In yet other embodiments of the invention, the nucleic acids of the invention comprise an ribonuclease-cleavable modified ribonucleotide residue.
The nucleic acids of the invention, including those for use as substrates in the methods of the invention, are at least 3 nucleotides in length, but are more preferably 5-30 nucleotides in length. In certain specific embodiments, the nucleic acids of the invention are 5-20, 5-15, 5-10, 7-20, 7-15 or 7-10 nucleotides in length.
In certain embodiments, the fluorescence-quenching group of the nucleic acids of the invention is 5xe2x80x2 to the cleavage domain and the fluorescence reporter group is 3xe2x80x2 to the cleavage domain. In a specific embodiment, the fluorescence-quenching group is at the 5xe2x80x2 terminus of the substrate. In another specific embodiment, the fluorescence reporter group is at the 3xe2x80x2 terminus of the substrate.
In certain embodiments, the fluorescence reporter group of the nucleic acids of the invention is 5xe2x80x2 to the cleavage domain and the fluorescence-quenching group is 3xe2x80x2 to the cleavage domain. In a specific embodiment, the fluorescence reporter group is at the 5xe2x80x2 terminus of the substrate. In another specific embodiment, the fluorescence-quenching group is at the 3xe2x80x2 terminus of the substrate.
In a preferred embodiments of the invention, a nucleic acid of the invention comprising the formula: 5xe2x80x2-N1-n-N2-3xe2x80x2, wherein: (a) xe2x80x9cN1xe2x80x9d represents zero to five 2xe2x80x2-modified ribonucleotide residues; (b) xe2x80x9cN2xe2x80x9d represents one to five 2xe2x80x2-modified ribonucleotide residues; and (c) xe2x80x9cnxe2x80x9d represents one to ten, more preferably four to ten unmodified ribonucleotide residues. In a certain specific embodiment, xe2x80x9cN1xe2x80x9d represents one to five 2xe2x80x2-modified ribonucleotide residues. In preferred modes of the embodiment, the fluorescence-quenching group or the fluorescent reporter group is attached to the 5xe2x80x2-terminal 2xe2x80x2-modified ribonucleotide residue of N1.
In the nucleic acids of the invention, including nucleic acids with the formula: 5xe2x80x2-N1-n-N2-3xe2x80x2, the fluorescence-quenching group can be 5xe2x80x2 to the cleavage domain and the fluorescence reporter group is 3xe2x80x2 to the cleavage domain; alternatively, the fluorescence reporter group is 5xe2x80x2 to the cleavage domain and the fluorescence-quenching group is 3xe2x80x2 to the cleavage domain.
In embodiments where a nucleic acid of the invention comprises the formula 5xe2x80x2-N1-n-N2-3xe2x80x2, the cleavage domain comprises the sequence xe2x80x9cauggcxe2x80x9d in a specific mode of such embodiments. In another specific mode of these embodiments, N1 and N2 each represent one 2xe2x80x2-modified ribonucleotide residue. The 2xe2x80x2-modified ribonucleotide residue is preferably an adenosine.
With respect to the kits of the invention, in addition to comprising a nucleic acid of the invention, the kits can further comprise one or more of the following: a ribonuclease; ribonuclease-free water, a buffer, and ribonuclease-free laboratory plasticware.